Baculovirus expression vectors have been successfully used to produce thousands of proteins for vaccines, therapeutics, and structure-function studies. Baculoviruses have also been adapted for use as mammalian expression systems and potential gene therapy agents. This proposal explores a novel strategy for purification, quantitation, and storage of high titer baculovirus stocks that are needed for mammalian transduction. Baculovirus infection of tissue culture cells is mediated by a form of virus that buds from cells. These viruses are highly infectious due to the presence of fusion proteins in their envelopes. Most of the replicative potential of the cell, however, is directed toward packaging of occlusion-derived viruses (ODV), which lack fusion peptides and have low infectivity in cell culture. The recent discovery of a peptide sequence that directs proteins to the envelope of ODV suggests that it should be possible to increase infectivity of these viruses. This would offer a number of advantages because ODV are not only more numerous, they are simpler to purify, concentrate and titer. ODV are also highly stable and do not lose titer over time, unlike budded virus. To test this idea, recombinant viruses will be produced with the baculovirus GP64 or VSV-G fusion protein linked to a sorting motif that targets heterologous proteins to ODV membranes. These recombinants will be screened for correct targeting of the fusion proteins and their infectivity relative to non-recombinant virus determined in vertebrate and invertebrate tissue culture systems. Experiments will also be conducted to discern whether Vankyrin-enhanced (VE) technology, currently marketed by ParaTechs as a method to increase baculovirus-mediated gene expression, would further boost yields of ODV and their infectivity. If successful, Phase II experiments would focus on producing an engineered viral backbone containing appropriate mammalian promoters and offering simple strategies for producing viral recombinants. Phase II would also expand the range of mammalian cells and explore animal models for gene therapy applications. This strategy of producing ODV that is highly infectious in tissue culture will add value to baculoviruses as mammalian expression vectors and gene therapy agents. The potential advantages offered with respect to simple concentration of viral stocks, rapid determination of titer, and long term storage without loss of infectivity represents a significant commercial opportunity and fully warrants undertaking this research. PUBLIC HEALTH RELEVANCE: Baculoviruses have been successfully adapted for use as mammalian expression systems;these BacMam vectors have allowed functional analyses of proteins not previously possible using other approaches. Whole viral genomes can also be delivered by baculovirus transduction, thus enabling the development of anti-viral drugs and examination of the mechanisms of drug resistance for significant human disease agents. This proposal seeks to develop new strategies for the production, purification, quantitation and storage of infectious baculovirus, which will add value to these expression systems.